The goal of this research effort is to understand how various types of white blood cells recognize and respond to the presence of a microorganism or cancer cell in the body, inappropriately recognize a normal component of the body (an auto-antigen), or deal with sterile tissue damage. Our experiments are designed to provide a detailed understanding of how the substances (antigens) making up microorganisms, cancer cells, or normal self-components, are made visible to the defending cells of the innate (anti-unspecific) or adaptive (antigen-specific) limbs of the host defense system and how recognition of infections, malignant cells, or adjuvants is linked through complex cell-cell interactions to the induction of protective or self-destruction effector responses. Previous work found that different dendritic cells initially present antigen to the majority of CD4 vs. CD8 T cells. Using a combination of technologies to identify each antigen-presenting cell type we showed last year that CD4 and CD8 T cells first engage distinct dendritic cells in an antigen-dependent manner, only later interacting with the same CD8, XCR1+ dendritic cell population, which serves as the platform by exchange of signals that augment the CD8 effector and/or memory response. This year, we have reported that plasmacytoid DC (pDC) contribute to orchestrating the formation of this XCR1+ DC-dependent co-operative platform within the lymph node. See also AI000758-20. We have also expanded our imaging technologies by developing methods that permit performing up to14 color fluorescent immunohistochemistry on lymphoid and other tissues and computationally analyzing the data to assign multiple stains to specific cells. This method previously allowed us to fully characterize all the migratory and resident dendritic cell subpopulations in skin draining lymph nodes of mice and to reveal the complex and heterogeneous, but non-random distribution of these various dendritic cell subsets. We call this entire set of analytic tools Histo-cytometry for its similarity in outcome to flow cytometric analysis of dissociated cell populations. Combining data from Histo-cytometry with 2P intravital imaging we identified last year a novel population of lymphatic sinus-resident dendritic cells in mouse lymph nodes and showed that these cells extend cytoplasmic processes directly into the subcapsular sinus to sample material in draining lymph. These cells are uniquely able to acquire particulate antigens accessing the lymph node by this route, with important implications for which dendritic cells present antigens delivered in particulate form. We also discovered that these sinus DC localize to the regions of the subcapsular sinus called interfollicular regions, where they replace the layer of CD169+ macrophages that are more restricted to the region over the primary B cell follicles, thus revealing a separation in antigen delivery function for T cells (sinus DCs) vs. B cells (sinus resident macrophages). This past year, we applied this method to a quantitative analysis of antigen delivery in lymph nodes, showing that even for small materials, penetration through conduits to deeper regions is quite limited. We found that DC position played a crucial role in the quantity of antigen acquired and that small changes in vaccine dose could selectively limit CD8 T cell activation, with important implications for design of vaccines geared toward elicitation of cell mediated immune responses. In a new application of Histo-cytometry, we developed methods for combining cell phenotyping with analysis of phospho-proteins, especially pSTAT molecules. We focused on the latter to address the question of the range over which cytokines act in vivo. While screening a panel of anti-pSTAT reagents, we noted an unexpected pattern of staining with antibody to pSTAT5. Further analysis showed that these small clusters of pSTAT5+ cells in the peripheral regions of all lymph nodes were Foxp3+ Tregs, that the pSTAT5 signal came from a central CD4 Teff cells making IL-2, that the clustered Tregs were especially rich in suppressive molecules like CTLA4 and CD73, that the TCR of Tregs was required for both the clustering phenomenon and effective suppression of Teff responses, and that IL-2 from the Teff was also required for optimal Treg suppression of responses. Most remarkably, the pSTAT5+ Treg clusters were of similar location and number on germfree mice. Together these data suggest that immune homeostatic is maintained not by Treg prevention of autoreactive T cell activation, but rather by a spatially localized feedback regulatory circuit in which activation of conventional T cells results in IL-2 production that other with physical clustering and signaling mediated by the TCR of Tregs, results in abrogation of the developing effector T cell response. These findings have changed the general view of Treg function from one in which they present autoreactive T cell responses to one in which they abort such responses, an important distinction and one that raises the question of what happens to the activated T cells when suppressed in this manner. See also AI000758-19. In a follow-on study, we employed this method to examine the distribution of Treg cells with a single TCR specificity and also collaborated with a computational group to show that the dimensionality of IL-2 signaling in vivo matched predictions from a computer model. This technology has also been applied to the analysis of signaling in the gut by innate lymphoid cells (ILCs) with findings that change how we understand the respective actions of CD4+ effector T cells vs. ILCs. To extend our Histo-cytometry to tissue volumes rather than sections, we have developed a novel tissue clearing method called Ce3D. This is simpler than existing methods, produces superior transparency, preserves fluorescent protein signals, and also allows multiplex staining with diverse fluorochrome labeled antibodies. We have developed a pipeline for 3D segmentation and shown that this method works in a variety of tissues. It promises to provide an unprecedented level of insight into cell organization, activity, and function in tissue samples, especially biopsies from patients, for example, those undergoing checkpoint blockade therapy in cancer. To extend our understanding of how immune cells navigate in 3D tissue spaces, we employed micro fabricated devices that permit precise control of chemoattractant gradient steepness and magnitude. Using these 3D systems, our findings suggest that temporal rather than spatial sensing plays a crucial role in persistent directional migration of various myeloid cell types to what are called guidance chemokines, and that the difference can be attributed to distinct ways in which negative feedback regulation operates with respect to the different classes of chemoattractants. We have developed a novel method for imaging the peritoneal wall, allowing us to study dynamically and in depth the roles of neutrophils, inflammatory monocytes/macrophages, and tissue resident macrophages in response to tumors, pathogens, and sterile injury. Remarkably, a focus on embryologically-derived fixed tissue macrophages revealed that they respond in a 2-phase manner to single cell sized sterile damage with a cloaking behavior that shields the damaged area from neutrophils and prevents the latter from swarming and increasing the lesion size. We suspect that this process is key to maintenance of tissue homeostasis in the face of episodic cell death and are designing studies to test this prediction. This new imaging platform can also be employed to analyze the effects of drugs and biologics on the innate and T cell responses to infection, tissue injury, or small tumor implants on the peritoneal wall.